1. Field of the Invention
This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. In particular, the invention relates to microminiaturization of genetic, biochemical and bioanalytic processes. Specifically, the present invention provides devices and methods for the performance of integrated and miniaturized sample preparation, nucleic acid amplification, and nucleic acid detection assays. These assays may be performed for a variety of purposes, including but not limited to forensics, life sciences research, and clinical and molecular diagnostics. The invention may be used on a variety of liquid samples of interest, including bacterial and cell cultures as well as whole blood and processed tissues. Methods for performing any of a wide variety of such microanalytical or microsynthetic processes using the Microsystems apparatus of the invention are also provided.
2. Background of the Related Art
Extraction and isolation of DNA from host cells is a cornerstone of modern molecular biology. One type of DNA, bacterial plasmid DNA has been particularly useful as a convenient vector for the insertion of genetic material into bacterial, yeast and mammalian cells. DNA isolated from an organism is inserted by being contiguously and covalently linked to plasmid DNA and is then introduced into a cell, such as a bacterial cell, and allowed to multiply, thereby creating large copy numbers of the plasmid in each cell. These plasmids may advantageously be harvested to provide a sufficient amount of DNA (typically on the order of several micrograms, although up to milligram quantities can be produced on an industrial scale) for a variety of experimental or therapeutic purposes. The harvesting of plasmid DNA, defined as its removal from cells and isolation from the genomic DNA content of the cells, has growing utility in life sciences research, diagnostics, therapeutics and other applications.
Currently, the extraction and isolation of DNA is either performed manually or through the use of robotic sample preparation stations. In either case, a variety of technologies and materials are used (see, for example, QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit Handbook, 1999, Qiagen GmbH, Max-Volmer-Strasse 4, 40724 Hildren, Germany; Birnboim and Doly, 1979, Nucl. Acids Res. 7: 1513-1522). Typically, cells are first incubated in a surfactant (detergent) solution, in some cases containing protein digesting enzymes such as Protease or Proteinase K. These lyse the cells, thereby releasing the DNA into solution. This is frequently performed under alkaline conditions, to destabilize nucleases and hydrolyze contaminating RNA. The DNA must then be separated from other cell constituents, which is performed using a number of different separation protocols, including, for example, selective precipitation of proteins and other cell debris, organic chemical extraction (using phenol and chloroform), and DNA affinity column chromatography. Plasmid DNA must also be isolated from contaminating cellular (bacterial genomic DNA). Filtration methods can produce a plasmid DNA solution, but the solutions required to solvate DNA are usually inappropriate for the desired final application of the DNA. As a consequence, plasmid DNA is removed from these solutions by ethanol precipitation, or solid-phase separation is used, which often requires further changes in solvent pH and salt concentration (especially for affinity binding methods using glass or silica). The technologies required for these steps include pipetting, pumping, filtration, washing, and centrifugation, requiring an expensive suite of devices and skilled operators thereof. The additional requirements of automated systems include sample transfer and robotics for the handling of sample containers.
This discussion illustrates the need in the art for more efficient, rapid, inexpensive automated methods and devices for performing DNA sample preparation, particularly plasmid DNA preparation.
In the field of integrated genetic analysis, some progress has been made in the integration of sample preparation, PCR, and detection via real-time fluorescence or hybridization methods (Anderson et al., 1998, xe2x80x9cAdvances in Integrated Genetic Analysis,xe2x80x9d in Proc. Micro Total Analysis ""98, Harrison and van den Berg, eds., Kluwer: Amsterdam, pp.11-16). These systems rely on macroscopic fluid handling systems such as pumps and valves that must be interfaced with the microfluidic devices within which fluids are processed.
However, there exists a need for devices and methods capable of processing cell cultures for harvesting DNA, particularly plasmid DNA.
In the biological and biochemical arts, analytical procedures frequently require incubation of biological samples and reaction mixtures at temperatures greater than ambient temperature. Moreover, many bioanalytical and biosynthetic techniques require incubation at more than one temperature, either sequentially or over the course of a reaction scheme or protocol.
One example of such a bioanalytical reaction is the polymerase chain reaction. The polymerase chain reaction (PCR) is a technique that permits amplification and detection of nucleic acid sequences. See U.S. Pat. Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis. This technique has a wide variety of biological applications, including for example, DNA sequence analysis, probe generation, cloning of nucleic acid sequences, site-directed mutagenesis, detection of genetic mutations, diagnoses of viral infections, molecular xe2x80x9cfingerprinting,xe2x80x9d and the monitoring of contaminating microorganisms in biological fluids and other sources. The polymerase chain reaction comprises repeated rounds, or cycles, of target denaturation, primer annealing, and polymerase-mediated extension; the reaction process yields an exponential amplification of a specific target sequence.
Methods for miniaturizing and automating PCR are desirable in a wide variety of analytical contexts, particularly under conditions where a large multiplicity of samples must be analyzed simultaneously or when there is a small amount of sample to be analyzed.
In addition to PCR, other in vitro amplification procedures, including ligase chain reaction as disclosed in U.S. Pat. No. 4,988,617 to Landegren and Hood, are known and advantageously used in the prior art. More generally, several important methods known in the biotechnology arts, such as nucleic acid hybridization and sequencing, are dependent upon changing the temperature of solutions containing sample molecules in a controlled fashion. Automation and miniaturization of the performance of these methods are desirable goals in the art.
Mechanical and automated fluid handling systems and instruments produced to perform automated PCR have been disclosed in the prior art.
U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Wilding et al. teach fluid handling on microscale analytical devices.
International Application, Publication No. WO93/22053, published Nov. 11, 1993 to University of Pennsylvania disclose microfabricated detection structures.
International Application, Publication No. WO93/22058, published Nov. 11, 1993 to University of Pennsylvania disclose microfabricated structures for performing polynucleotide amplification.
Wilding et al., 1994, Clin. Chem. 40: 43-47 disclose manipulation of fluids on straight channels micromachined into silicon.
Kopp et al., 1998, Science 280: 1046 discloses microchips for performing in vitro amplification reactions using alternating regions of different temperature.
One drawback of the synthetic microchips disclosed in the prior art for performing PCR and other temperature-dependent bioanalytic reactions has been the difficulty in designing systems for moving fluids on the microchips through channels and reservoirs having diameters in the 10-100 xcexcm range. This is due in part to the need for high-pressure pumping means for moving fluid through the small sizes of the components of these microchips. These disabilities of the prior art microchips limits the usefulness of these devices for miniaturizing and automating PCR and other bioanalytic processes.
Thus, there exists a need in the art for devices and methods that provide integrated sample preparation and analysis, particularly of DNA samples. This need is particularly acute for high throughput analyses, which are currently burdened by the high costs and complexity of automated, typically robotic, systems. Integration of DNA sample preparation and analysis would be particularly useful if it reduced the current need in the art for need for multiple, complex technologies that demand highly-skilled operators. Importantly, for DNA analysis integration of sample preparation and in vitro amplification methods would minimize the possibility of contamination and sample carry-over, which is particularly important in high-sensitivity techniques such as various in vitro amplification reactions used in the art.
Some of the present inventors have developed a microsystem platform and a micromanipulation device to manipulate said platform by rotation, thereby utilizing the centripetal forces resulting from rotation of the platform to motivate fluid movement through microchannels embedded in the microplatform, as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996, now U.S Pat. No. 6,319,469; 08/910,726, filed Aug. 12, 1997, now U.S. Pat. No. 6,143,248; 08/995,056, filed Dec. 19, 1997, now U.S. Pat. No 6,143,147; 09/315,114, filed May 19, 1999; 09/579,492, filed May 12, 2000 and 09/595,239 filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein.
This invention provides Microsystems platforms as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; 09/315,114, filed May 19, 1999; 09/579,492, filed May 12, 2000 and 09/595,239, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein.
The invention provides apparatus and methods for performing microscale processes on a microplatform, whereby fluid is moved on the platform in defined channels motivated by centripetal force arising from rotation of the platform. The Microsystems platform is provided to perform integrated and miniaturized sample preparation, nucleic acid amplification, and nucleic acid detection assays. A first element of the apparatus of the invention is a microplatform that is a rotatable structure, most preferably a disk, the disk comprising fluid (sample) inlet ports, fluidic microchannels, reagent reservoirs, collection chambers, detection chambers and sample outlet ports, generically termed xe2x80x9cmicrofluidic structuresxe2x80x9d. The disk is rotated at speeds from about 1 to about 30,000 rpm for generating centripetal acceleration that enables fluid movement through the microfluidic structures of the platform. The disks of the invention also preferably comprise air outlet ports and air displacement channels. The air outlet ports and in particular the air displacement ports provide a means for fluids to displace air, thus ensuring uninhibited movement of fluids on the disk. The disk, and most preferably a face of the platform, may also contain heating elements for raising the temperature of fluids contained therein to temperatures greater than ambient temperatures. Specific sites on the disk also preferably comprise elements that allow fluids to be analyzed.
A preferred embodiment of the platforms of the invention is a platen that rotates with the microfluidics disk. The platen is most preferably a printed circuit board comprising resistive heating elements, thermoelectric (Peltier) elements, temperature sensors, assay optics and microprocessor and other electronic components. Electrical communication between a rotating platen and stationary power sources, motor controllers, temperature controllers, and computers is most preferably accomplished through a slip-ring assembly. By mounting the microfluidic disk on the platen and rotating both disk and platen together, the distribution and flow rate of fluid throughout the microfluidic structures as well as the temperature of fluid within localized regions of the microfluidics disc can be controlled.
In a preferred embodiment, one face of the microfluidics disk is mounted onto a face of the platen and the temperature of fluids at particular positions within the microfluidics disk is controlled through temperature exchange between the platen and disk. In alternative embodiments, a microfluidic disk is positioned between two platens, each comprising elements that effect temperature exchange between the disk and thermal regulation elements comprising the platens. In a preferred embodiment, the platen is a printed circuit board with resistive heating elements, Peltier elements and temperature sensors embedded therein or affixed thereto. In an alternative embodiment, thermal regulation within the microfluidic disk is achieved by permanently bonding a layer comprising resistive heaters directly to the disk; in this case, fluids within the disk are heated to temperatures greater than ambient temperature with resistive heating elements and cooled to temperatures above or equal to ambient temperature by spinning the disk and through the loss of heat to the environment. As with the platen, electrical communication between this composite disk and power supplies, temperature controllers and computers is most preferably accomplished through a slip-ring assembly.
In a first aspect, the present invention provides devices and methods for the performance of integrated and miniaturized sample preparation for the extraction, isolation, and purification of DNA from cells. In preferred embodiments, the devices and methods of the invention are particularly provided to isolate plasmid DNA from bacterial cells.
The plasmid DNA sample preparation platforms of the invention are provided to perform the following functions: sample processing to free DNA from the bacterial cell; filtration of the resultant solution to remove bacterial cell fragments; application of the solution to a binding matrix using solvent conditions that promote DNA binding to the matrix; washing of bound DNA and replacement of the original solution by a solution that is compatible with further analytical methods; and elution of the DNA from the binding matrix in a suitable solvent. The DNA thus eluted can be isolated, amplified in vitro or sequenced using methods known in the art. The platforms of the invention are provided comprising microfluidic structures that perform plasmid DNA sample preparation as described in further detail below. These microstructures are illustrated for clarity with regard to a single microstructure. However, platforms comprising a multiplicity of such plasmid DNA preparation microfluidic structures are provided by the invention, wherein the microfluidics structures are arrayed on the surface of the platform with a density determined by the size of the platform and the volumetric capacity of the chambers and reservoirs comprising the microfluidic structures as disclosed herein.
In a second aspect, the invention is provided having microfluidics structures as described herein for performing an integrated suite of biochemical processes for accomplishing in vitro amplification reactions. These include sample processing to isolate DNA from bacterial or mammalian cells; sample conditioning to adjust the solution conditions to those appropriate for PCR; mixing of the conditioned sample with PCR reagents, including deoxyribosenuclotides, polymerase enzyme, primers, and appropriate salts, buffers and additives; and thermal cycling to effect PCR.
In certain preferred embodiments, the discs of the invention are provided with a multiplicity of microfluidics structures that enable to platform to process and amplify several samples simultaneously. In these embodiments, multiple copies of an arrangement of microfluidics structures for performing the biochemical reaction suite are arrayed on the disc, and sample input ports or reservoirs provided for each copy, thereby permitting processing of multiple samples. In addition, the portion of the sample DNA to be amplified can be independently, by the choice of amplification primers provided in each of the individual copies of the microfluidics structures arrayed on the disc, thereby permitting amplification xe2x80x9cmultiplexingxe2x80x9d of a particular sample. Alternatively, the same primers can be provided to process in parallel multiple samples for amplification of the same target fragment in the DNA of each sample. Independent thermal cycling profiles, including the temperature used for each step of the amplification cycle, temperature ramp-rates, and hold times, may be individually programmed into the instrument for each of the microfluidics structures or for each of the samples processed.
The invention advantageously permits simultaneous, independent thermal cycling of a multiplicity of different samples, independent amplification of different target fragments from a particular sample, or both. This feature also enables a user to optimize thermal cycling parameters for a single sample or amplicon quickly and in a single experiment, by varying reaction parameters on a plurality of the microfluidics structures arrayed in the disc, thereby simultaneously performing multiple experiments simultaneously. Since particular copies of the microfluidics structures can be arranged in microfluidic isolation from other copies on the platform, portions comprising less than all of the microfluidics structures can be discretely used and the remainder retained for future use.
In alternative embodiments of the platforms of the invention, metering structures as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000 and incorporated by reference herein, are used to distribute aliquots of reagent to each of a multiplicity of mixing structures, each mixing structure being fluidly connected to one of a multiplicity of sample reservoirs, thereby permitting parallel processing and mixing of the samples with a common reagent. This reduces the need for automated reagent distribution mechanisms, reduces the amount of time required for reagent dispensing (that can be performed in parallel with distribution of reagent to a multiplicity of reaction chambers), and permits delivery of small (nL-to-xcexcL) volumes without using externally-applied electromotive means.
The assembly of a multiplicity of collection chambers on the platforms of the invention also permits simplified detectors to be used, whereby each individual collection/detection chamber can be scanned using mechanisms well-developed in the art for use with, for example, CD-ROM technology. Finally, the platforms of the invention are advantageously provided with sample and reagent entry ports for filling with samples and reagents, respectively, that can be adapted to liquid delivery means known in the art (such as micropipettors).
The discs of this invention have several advantages over those that exist in the centrifugal analyzer art. Foremost is the fact that flow is laminar due to the small dimensions of the fluid channels; this allows for better control of processes such as mixing and washing. Secondly, the small dimensions conferred by microfabrication enable the use of xe2x80x9cpassivexe2x80x9d valuing, dependent upon capillary forces, over much wider range of rotational velocities and with greater reliability than in more macroscopic systems. To this are added the already described advantages of miniaturization.
The present invention solves problems in the current art through the use of a microfluidic disc in which centripetal acceleration is used to move fluids. It is an advantage of the microfluidics platforms of the present invention that the fluid-containing components are constructed to contain a small volume, thus reducing reagent costs, reaction times and the amount of biological material required to perform an assay. It is also an advantage that the fluid-containing components are sealed, thus eliminating experimental error due to differential evaporation of different fluids and the resulting changes in reagent concentration. Because the microfluidic devices of the invention are completely enclosed, both evaporation and optical distortion are reduced to negligible levels. The platforms of the invention also advantageously permit xe2x80x9cpassivexe2x80x9d mixing and valving, i.e., mixing and valving are performed as a consequence of the structural arrangements of the components on the platforms (such as shape, length, position on the platform surface relative to the axis of rotation, and surface properties of the interior surfaces of the components, such as wettability as discussed below), and the dynamics of platform rotation (speed, acceleration, direction and change-of-direction), and permit control of assay timing and reagent delivery.
The devices of the invention also implement simpler, more robust, and more economical sample preparation for performing in vitro amplification reactions such as PCR. All mechanical aspects of sample processing are carried out using a single motor that rotates the disc at prescribed velocities, thereby driving fluids on the disc through microchannels and other microfluidics structures. This is in advantageous over current sample preparation methods involving robotic pipetting stations or other fluid transfer mechanisms, automation for the delivery of processing plates to different xe2x80x9cstations,xe2x80x9d or both.
The invention advantageously integrates sample preparation with thermal cycling for PCR, thereby eliminating additional fluid transfer steps. This minimizes the potential for contamination or fluid loss.
The platforms of the invention reduce the demands on automation in at least three ways. First, the need for precise metering of delivered fluids is relaxed through the use of on-disc metering structures, as described more fully in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; 09/315,114, filed May 19, 1999; 09/579,492, filed May 12, 2000 and 09/595,239, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. By loading imprecise volumes, slightly in excess of those needed for the assay, and allowing the rotation of the disc and use of appropriate microfluidic structures to meter the fluids, much simpler (and less expensive) fluid delivery technology may be employed than is the conventionally required for high-density microtitre plate assays.
Second, the total number of fluid xe2x80x9cdeliveryxe2x80x9d events on the microfluidic platform is reduced relative to microtiter plates. By using microfluidic structures that sub-divide and aliquot common reagents (such as reagent solutions, buffers, and enzyme substrates) used in all assays performed on the platform, the number of manual or automated pipetting steps are reduced by at least half (depending on the complexity of the assay). A reduction in fluid transfers to the device can reduce total assay time. Examples of these structures have been disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and incorporated by reference herein.
The invention also provides on-platform means for mixing reagents with sample and washing the resulting reaction products, removing the need for transferring the assay collection chamber(s) to a separate xe2x80x9cwashxe2x80x9d station. This also reduces manipulation of the assay device as well as providing controlled and integrated fluid processing.
The invention disclosed herein is flexible as to sample and source, being capable of isolating nucleic acid from bacteria, whole animal blood, tissues and cellular sources. It is rapid, being about 50% more rapid than existing xe2x80x9cautomatedxe2x80x9d nucleic acid preparatory methods. The nucleic acid output of the system is of a quality higher than or equal to methods known in the art. The system is simple and easy to use, robust because it is not dependent on operator variability. In addition, the platforms and systems disclosed are self-contained and integrated, thereby minimizing both operator handling and error.